Proceedings of the 2nd IEEE InternationalConference on Nano/Micro Engineered and Molecular Systems

January 16 - 19, 2007, Bangkok, Thailand

Chitosan Clad Manganese Doped Zinc SuiphideNanoparticles as Biological Labels

Hemant C. Warad 1, Chanchana Thanachayanont 2, Gamolwan Tumcharern 3, and Joydeep Dutta '^lCentre of Excellence in Nanotechnology, School of Engineering and Technology, Asian Institute of Technology, Thailand.

2 National Metal and Materials Technology Center, Thailand.3 National Nanotechnology Center, Thailand

Abstract- Here we report the synthesis in aqueous media ofredispersible zinc sulphide quantum dots doped with manganese,capped by biocompatible 'chitosan', molecules. Thenanoparticles show highly efficient luminescence with a peak ataround 590 nm that has been correlated to the manganesedopants. The synthesis involves very simple precipitationtechniques that may lead to the development of a cost effectivealternative of using nanocrystals to replace fluorophores forbiological staining. The biocompatible capping agent, chitosan,renders the particles suitable for bio-labels (markers). The 40 nmsized spherical nanoparticles consists of multiple crystallites ofabout 4 - 5 nm as observed in High Resolution TransmissionElectron Microscopy studies.

Keywords-Colloid, Fluorescence, Marker, Quantum Dot, ZincCompound

I. INTRODUCTION

Biomolecules labeled with luminescent colloidalsemiconductor quantum dots have potential applications influoro-immunoassays and biological imaging [1, 2]. Quantumdots (QDs) have several advantages over conventional organicfluorophores as they are more efficient in luminescencecompared to organic dyes and their emission spectra arenarrow, symmetric and tunable according to the particle sizesand material composition which show excellent photostability.Due to their broad absorption spectra they can be excited to allcolors of the QDs by a single excitation light source [3]. Alsothe excitation and emission peaks are easily distinguishable asexcitation can be done in any wavelength in the broadabsorption band [4]. Due to inherent advantages of QDs overorganic dyes it could allow the integration of nanotechnologyand biotechnology leading to major advances in medicaldiagnostics, molecular biology and cell biology [5, 6].

The utility of QDs to sense biological surfaces dependcritically on QDs surface properties and surfacefunctionalization. For most biological applications,biocompatible polymers like Polyvinvy Alcohol or proteinslike lysine, streptavidin, biotin etc. have been used [7, 8].Proteins are expensive but offer attractive possibilities forbiological applications. In this work we have used chitosan forsteric stabilization of the synthesized colloids that ineffect alsoform the biocompatible coating around ZnS nanoparticles.

Chitosan is an inexpensive, biocompatible, biodegradablebiopolymer that has free amine groups which serve asfunctional elements for biological applications [9]. Chitosan isa partially deacetylated polymer of acetyl glucosamine

(2 acetamido-2-deoxy b-1, 4-D-glucan) commonly known aschitin, which is found in a wide range of natural sources(crustaceans, fungi, insects, annelids, molluscs etc.). It isinsoluble in alkaline, strong acids and in neutral pH solutionsdue to its rigid crystalline structure and intra and intermolecular hydrogen bonding network [10], but is highlysoluble in dilute acids. The dissolution of chitosan in diluteacidic solution, results in the protonation of amine groups(-NH2) with the formation of positively charged NH3,imparting a polycationic nature to the polymer, that has beenutilized for polyelectrolyte applications [11]. The amine groupsof chitosan are the major effective binding sites for metal ionsforming stable complexes by co-ordination, which is of interestwhen used as capping agent for nanoparticles [12, 13]. Thisproperty makes chitosan to be used as a flocculent, gas-selective membrane, plant disease resistance promoter, anti-cancer agent, wound healing promoting agent, andantimicrobial agent [14 - 20]. Long chain chitosan with itslarge number of cationic amine groups form multiple bindingsites with metal particle surfaces. This effectively encapsulatesthe particle, providing steric hindrance from agglomeration ofthe particles in a colloidal system. This inherent property ofchitosan makes it an interesting polymer to be used for cappingnanoparticles to avoid agglomeration. The unattached aminescan allow the possibility of being functionalized for differenttargeting purposes.

Cytotoxicity of chitosan coated ZnS nanoparticles has alsobeen studied and will be reported. We will discuss thesynthesis of ZnS:Mn2+ nanophosphors and discuss about thephotoluminescence properties of the QDs. We will also presentthe preliminary studies on the attachment of ZnS nanoparticlesto fibroblast cells and to anionic cell walls of Bacillus cereous.

II. EXPERIMENTAL SECTIONThe inorganic wet chemical synthesis used to prepare

ZnS:Mn2+ nanocrystals passivated with chitosan is performedat room temperature and under ambient conditions. Mediummolecular weight chitosan with a 75 - 85 % degreedeacetylation were obtained from Sigma-Aldrich. Zinc acetate,manganese acetate and sodium sulphide were purchased fromUnivar, Fluka Chemika and Panreac respectively. Dilute aceticacid used to dissolve chitosan polymer was prepared fromglacial acetic acid obtained from Sigma-Aldrich.

Stock solutions of 0.25 M zinc acetate and 0.25 M sodiumsulphide were prepared by dissolving 2.74 g and 3.00 g ofrespective salts in 50 ml of de-ionized water. 5 mM stock

*Contact author phone: +66-2-524-5680; fax: +66-2-524-5697; e-mail:joygait.ac.th

1-4244-0610-2/07/$20.00 C)2007 IEEE 342

solution of manganese acetate was prepared by dissolving61.27 mg of the salt in 50 ml of de-ionized water. Stocksolution of 0.1 0% polymer was prepared by dissolving 100 mgof chitosan in 100 ml of dilute acetic acid (1 % concentration).To synthesize nanoparticles of ZnS:Mn2+ , 40 ml de-ionizedwater was heated to boil. To this 1 ml of the above preparedzinc acetate and manganese acetate was added and stirred withcontinuous heating. Upon boiling, 7 ml of 0.1 % chitosansolution was then added with continuous stirring and heatingfollowing which 1 ml of sodium sulphide was added to thesolution. Upon the addition of sodium sulphide a whiteprecipitate of ZnS nanoparticles is formed instantaneously andthe solution is rapidly cooled in ice.

A pH of around 5.4 results during the synthesis ofZnS:Mn2+ nanoparticles. The supernatant was then centrifugedat 4000 rpm for 20 minutes to sediment agglomerated particles.To the colloid, dilute acetic acid was added to remove excesschitosan hanging from the particles. The supernatant was thendialyzed to remove other un-reacted ions. In some of theexperiments (e.g. for X-Ray diffraction studies), after washingthe colloid several times with de-ionized water, the sample wasfreeze dried to obtain fine powder ofZnS:Mn2+ nanoparticles.

The morphology of the nanoparticle were studied in aJEOL, JSM-6301F scanning electron microscope (SEM)operating with primary electron energy of 20 KeV. Highresolution electron microscopy (HRTEM) was carried out in aJEOL, JEM-210OF transmission electron microscope (TEM)operating at a source voltage of 200 kV. The TEM sample wasprepared by dispersing the sample in dilute acetic acid (dilutedthrice) and dropping it onto a lacy holey carbon coated coppergrid. X-ray diffraction (XRD) patterns were recorded on aJEOL, JDX-3530 diffractometer using Cu-radiation (Cu Ka,wavelength = 1.540600A). UV-Visible absorption spectra wereobtained with an Elico spectrophotometer SL164 and thephotoluminescence and excitation spectra with a Perkin-ElmerLS 50B spectrometer. Photo correlation spectra measurementswere done in a Zetasizer Nano ZS Zen-3600 from MalvernInstruments.

III. RESULTS AND DISCUSSIONSThe general sequence of the formation of Zinc Sulphide

nanoparticles in co-precipitation techniques proceed bynucleation, growth and Ostwald ripening followed byagglomeration or aggregation that are dominated by the surfaceenergy of the nanoparticles [21]. Here, manganese doped zincsulphide (ZnS:Mn ) nanoparticles were synthesized from ahomogenous solution of zinc acetate that was reacted withsodium sulphide in the presence of manganese acetate inaqueous media.

Zinc acetate in solution (de-ionized water in our case)dissociates into zinc (Zn2+) and Acetate (Ac-) ions. Whensodium sulphide (which itself dissociates into sodium [Na+]and sulphide [S2-] ions) is added to the solution containing theZn2+ ions, the S2- reacts with the Zn2+ ions forming zincsulphide (ZnS). The Na+ ions present in the solution plays avery important role in deciding the size of the ZnSnanocrystals. The manganese (Mn2 ) ions which are present insmall quantities compared to the Zn2+ ions (1/100th the

concentration of Zn2+ ions), replace some of the Zn2+ in theZnS lattice thereby doping the ZnS which gives ZnS itscharacteristic Orange red luminescence (590 nm).

Nanoparticles have a strong tendency to agglomerate andsettle down due to their Van der Waal's interactions. To avoidagglomeration of nanoparticles a repulsive force must be addedbetween the particles to balance the attraction process.Chitosan exhibits polycationic structure, inducing sterichindrances to show net positive charge due to the presence ofthe amine groups. The key idea behind this work is, cappingthe surface of the ZnS nanoparticles with chitosan so that thenet unattached positive amine groups will decorate thenanoparticles surface, serving to functionalize them.

Figure 1. SEM micrograph of ZnS:Mn21 nanoparticles showing averageagglomerate sizes of around 30-40 nm

A typical Scanning Electron Micrograph of ZnS:Mn2+nanoparticles are shown in figure 1. SEM study shows that theparticles have smooth surfaces due to the surface passivation ofthe luminescent nanocrystals by chitosan polymer. We observethat the agglomerate sizes are around 30 - 40 nm. Thestabilizing agent passivates the surface of the particle as hasbeen further confirmed from infrared spectroscopy (FTIR).

60

50

40

30

20

10

ClCoCo

Ox00

CW

Wavenumber (cm'1)CoOC

Figure 2. FTIR spectra of vigerously washed chitosan capped ZnS:Mn2.Peaks at 1560 cm-1 and 659 cm-1 are representative of amine group present

sterically capping the nanoparticles.

It is clear from the spectra (figure 2) that the particles arecapped with chitosan that are indicated by the presence ofstrong peak at 1560 cm-' and weak one at 659 cm-', that areindicative of the presence of amine groups. The presence ofamine group even after rigorous washing of the colloidalnanoparticles suggests further that chitosan enrobes the

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nanoparticles preventing agglomeration of particles by sterichindrance [22].

ZnS has a direct band gap of 3.6 eV with an effectiveenergy band gap of 339 nm at room temperature. Upon dopingzinc sulphide with manganese, the Mn2+ ions substitute theZn2+ ions in the ZnS crystal acting as trap sites for the electronsand holes. An electron can undergo photo-excitation process inthe host ZnS lattice of nanoparticles and subsequently decayvia a non-radiative transition to the 4T, level to the 6Al level[24]. The strong emission could be attributed to the radiativedecay between these localized states of manganese inside theZnS bandgap [21].

Figure 3. HRTEM reveals the individual grain boundaries. Approximatecrystallite sizes are 4-5 nm

High Resolution Transmission Electron Microscopyanalysis reveals that the 40 nm agglomerates arepolycrystalline having an average crystallite of approximately4 - 5 nm as shown in figure 3. This is reasonably consistentwith the estimation of the crystallite sizes obtained from theX-ray powder diffractogram (XRD) analysis [23].

Figure 4 shows the XRD of nanocrystalline ZnS:Mn2+ thatreveal the quantum dots of ZnS:Mn2+ have zinc blendestructure with planes at { 111 }, {220} and {3 11 } respectively.The nanocrystals have lesser lattice planes compared to bulkwhich contributes to the broadening of the peaks in thediffraction pattern. This broadening of the peak could also arisedue to the micro-straining of the crystal structure arising fromdefects like dislocation and twinning etc. These defects arebelieved to be associated with the chemically synthesizednanocrystals as they grow spontaneously during chemicalreaction since chemical species get very little time to diffuse toan energetically favorable site. It could also arise due to lack ofsufficient energy needed by an atom to move to a proper site informing the crystallite. The crystallite size determined from theDebye-Scherrer formula from the major peak (from { 111 }plane) centered at 20 = 28.520 was established to be about 1.97nm.

340 {111}

t 265

{2 2 O}v, 190

V 115

4020 30 40

Angle (20)

{3 1 1}

50

Figure 4. XRD scan ofZnS:Mn21 nanoparticle showing broadening of thepeaks at { 111 }, {220} and {3 11 } due to nanocrystalline nature of the crystal.

These quantum dots have zinc blende structure. The crystallite size ascalculated by Debye-Scherrer formula is 1.97 nm.

0.8

4 0.6

- 0.4

0.2

O275 375 475 575 675

Wavelength (nm)

Figure 5. Normalized UV-Vis absorption and fluorescence emission spectraof ZnS:Mn2+ These nanoparticles have an optical edge at 285 nm and with a

strong emission at 590 nm. Emission peak at 424 nm is from ZnSnanocrystallites.

The colloid containing ZnS:Mn2+ nanoparticles have anoptical absorption edge at around 265 nm and under ultraviolet(UV) exposure (figure 5), it glows with an orange-red color.The strong emission peaking at 590 nm is attributed toradiative decay between localized states of manganese withinZnS bandgap. The emission peaking around 425 nm is typicalluminescence of undoped ZnS resulting from the transition ofelectrons from shallow states near the conduction band tosulphur vacancies present near the valence band.

Conventional methods of detecting microbialcontamination have rely on time-consuming steps, followed bybiochemical identification, having a total assay time of up to1 week in certain cases [25]. A great deal of research has beenfocused on the development of biological sensors for thedetection of microorganisms, allowing rapid and "real-time"identification [26].

Most bacteria range from 0.2 - 2.0 ptm in breadth and about2 - 8 pm in length. The basic shapes of bacteria vary fromspherical coccus, rod shapes bacillus and spiral. Since the cellwalls of bacilli are anionic in nature, any functionalizedmaterial having a net positive charge (for example luminescentnanocrystals capped with chitosan at slightly acidic pH) willattach and organize on the cell walls leading to biolabeling.

In a simple experiment to demonstrate that the luminescentnanocrystals attach to the walls of the bacillus, some culturedbacilli were fixed on to a glass slide. Freeze dried powder ofZnS: Mn2+ was sprinkled on the surface of the microorganismsand the slide was rinsed with deionized water to wash awayunattached nanoparticles. Upon observation under a fluorescentmicroscope, distinct orange red glow was observed which took

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the shape of the bacilli, (figure 6). As a control experiment,some of the colloidal suspension of the nanocrystals weresmeared on to the glass slide (without the bacilli) and heated.After vigorous washing, no luminescence was observed,conclusively proving that the luminescent nanocrystals indeedattach strongly to the cell wall of the bacilli.

Figure 6. Chitosan capped ZnS: Mn2+ with bacillus as seen under aFluorescence Microscope. The orange glow is from bio-functionalized

ZnS:Mn2+attached to bacillus.

Though no specific experiments were conducted to proveselective attachment of the chitosan capped ZnS:Mn2+nanocrystals to specific species of bacteria, initial results showthat these nanocrystals preferentially attach to gram-positivebacteria. Current focus of our research is to modify the cappingagent of the nanoparticles to enhance specificity.

Cytocompatibility of the nanoparticles, were studied byadding colloidal ZnS:Mn2+ nanoparticles to a culture dishcontaining human 3T3 fibroblast cells. The cells were observedunder a microscope over intervals of time. It was observed thatthe cells did not perish even after 8 days. It was also noticedthat the growth of new cells were inhibited by the presence ofchitosan, which is a known bactericidal agent [27]. Thebiopolymer chitosan, as capping agent makes thesenanoparticles cytocompatible, allowing them to be used as bio-labels.

CONCLUSION

Synthesis of colloidal suspensions of zinc sulphide dopedwith manganese was achieved from a simple precipitationreaction leading to particle sizes of 30 - 40 nm consisting ofpolycrystalline consisting of crystallites of 4 - 5 nm. Theparticles were stabilized in the colloidal suspension with abiocompatible polymer 'chitosan'. It was found that chitosanprovides an effective passivation of the unsaturated bonds onthe particle surfaces, eliminating the non-radiative pathwaysfor the excited electrons, thus leading to a high luminescenceefficiency of the ZnS:Mn2+ nanoparticles. ZnS:Mn2+ has zincblende structure, as observed by X-ray diffraction, and has acrystallite size of 1.97 nm as calculated by Debye-Scherrerformula. Under UV exposure, the colloidal suspension orpowder of ZnS:Mn2+ glows with an orange-red luminescencepeaking at around 590 nm.

The biopolymer chitosan, as capping agent makes thesenanoparticles biocompatible, allowing them to be used asbio-labels. We have demonstrated that the chitosan capped

ZnS nanoparticles attaches to bacillus and upon irradiation withUV light glows with an orange color.

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